Methods of growing tumor infiltrating lymphocytes in gas-permeable containers

ABSTRACT

An embodiment of the invention provides a method of promoting regression of cancer in a mammal comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium therein; obtaining tumor infiltrating lymphocytes (TIL) from the tumor tissue sample; expanding the number of TIL in a second gas permeable container containing cell medium therein using irradiated allogeneic feeder cells and/or irradiated autologous feeder cells; and administering the expanded number of TIL to the mammal. Methods of obtaining an expanded number of TIL from a mammal for adoptive cell immunotherapy are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/466,200, filed Mar. 22, 2011, which is incorporatedby reference in its entirety herein.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL)can lead to positive, objective, and durable responses in cancerpatients. However, this therapy can involve sophisticated cellprocessing techniques and equipment. These procedures have introducedtechnical, regulatory, and logistic challenges to the successful use ofTIL as a biological therapy. Accordingly, there is a need in the art forimproved methods for growing TIL for use in adoptive cell therapy.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of promoting regressionof cancer in a mammal comprising obtaining a tumor tissue sample fromthe mammal; culturing the tumor tissue sample in a first gas permeablecontainer containing cell medium therein; obtaining tumor infiltratinglymphocytes (TIL) from the tumor tissue sample; expanding the number ofTIL in a second gas permeable container containing cell medium thereinusing irradiated allogeneic feeder cells and/or irradiated autologousfeeder cells; and administering the expanded number of TIL to themammal.

Another embodiment of the invention provides a method of obtaining anexpanded number of TIL from a mammal for adoptive cell immunotherapycomprising obtaining a tumor tissue sample from the mammal; culturingthe tumor tissue sample in a first gas permeable container containingcell medium therein; obtaining TIL from the tumor tissue sample;expanding the number of TIL in a second gas permeable containercontaining cell medium therein using irradiated allogeneic feeder cellsand/or irradiated autologous feeder cells.

Still another embodiment of the invention provides a method of obtainingan expanded number of TIL from a mammal for adoptive cell immunotherapycomprising obtaining a tumor tissue sample from the mammal; obtainingTIL from the tumor tissue sample; expanding the number of TIL in a gaspermeable container containing cell medium therein using irradiatedallogeneic feeder cells and/or irradiated autologous feeder cells.

Another embodiment of the invention provides a method of promotingregression of cancer in a mammal comprising obtaining a tumor tissuesample from the mammal; obtaining TIL from the tumor tissue sample;expanding the number of TIL in a gas permeable container containing cellmedium therein using irradiated allogeneic feeder cells and/orirradiated autologous feeder cells; and administering the expandednumber of TIL to the mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a graph showing the numbers of TIL produced by 10 tumorfragments from eight tumor samples in 24-well plates (diamonds) andG-Rex10 flasks (squares). For each individual tumor sample, 10 fragmentswere seeded into a 24-well plate at 1 piece per well and 10 fragmentswere seeded into a single G-Rex 10 flask. Cells were harvested by 7 to23 days of culture, pooled if collected from 24-well plate, and counted.A total of 8 samples were tested.

FIG. 1B is a graph showing the number of TIL produced per each tumorfragment by 7 to 23 days of culture of 5, 10, 20 and 30 tumor fragmentsin G-Rex10 flasks. Because only G-Rex10 flasks with 10 fragments wereused in all experiments, the data were normalized using the number ofcells produced in G-Rex10 flasks with 10 fragments. The number of TILproduced in each flask was divided by the number of fragments in theflask and this value was divided by the number of TIL produced inG-Rex10 flasks with 10 fragments from the same patient divided by 10.The average number of TIL produced by each tumor fragment in G-Rex 10flasks seeded with 10 fragments was 7.51×10⁶ cells per fragment (n=11).

FIG. 1C is a graph showing the total number of TIL produced by 7 to 23days of culture of 5, 10, 20 and 30 tumor fragments in G-Rex10 flasks.The data were normalized using the number of cells produced in G-Rex10flasks with 10 fragments. The total number TIL produced in each G-Rex10flask was divided by the number of TIL produced by each G-Rex10 flaskseeded with 10 tumor fragments from the same patient. The average numberof TIL produced by G-Rex10 flasks seeded with 10 tumor fragments was75.1×10⁶ (n=11).

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a method of promoting regressionof cancer in a mammal comprising obtaining a tumor tissue sample fromthe mammal; culturing the tumor tissue sample in a first gas permeablecontainer containing cell medium therein; obtaining tumor infiltratinglymphocytes (TIL) from the tumor tissue sample; expanding the number ofTIL in a second gas permeable container containing cell medium thereinusing irradiated allogeneic feeder cells and/or irradiated autologousfeeder cells; and administering the expanded number of TIL to themammal.

The inventive methods provide numerous advantages. For example, methodsof promoting regression of cancer and obtaining an expanded number ofTIL using gas permeable containers are simpler, less labor-intensive,use less reagents, and can be performed using simpler equipment thanprocedures using non-gas permeable containers (e.g., T-flasks (e.g.,T-175 flasks), bags, and multi-well plates). In addition, gas permeablecontainers may advantageously protect the cells from microbialcontamination more effectively than non-gas permeable containers whichmay be “open” systems. In addition, methods using gas permeablecontainers may advantageously reduce the number of containers that areused compared to methods using non-gas permeable containers, therebyreducing the amount of labor necessary to carry out the methods and alsoreducing the risk of microbial contamination. Thus, producing cells ingas permeable containers may be more suitable for compliance with thecurrent manufacturing practice (cGMP) conditions that are required for,e.g., Phase III clinical trials. Moreover, methods using gas-permeablecontainers advantageously reduce the final culture volume to lower thanthat obtained with non-gas permeable containers, which advantageouslylowers the incubator capacity required to grow the cells, reduces theamount of reagents (e.g., cell culture medium and additives) necessaryto grow the cells, and simplifies the equipment and/or procedures forconcentrating and washing the cells. Another advantage of the inventivemethods is that the cells may be fed less frequently in gas-permeablecontainers (e.g., about every three to four days) than in non-gaspermeable containers (e.g., every other day), particularly when thecells and/or tumor tissue sample are cultured submerged under at leastabout 1.3 cm of cell culture medium in a gas permeable container.Moreover, cells in gas permeable containers may be handled lessfrequently than cells in non-gas permeable containers (e.g., bags),which may minimize disturbance of the tumor fragment and provide morereproducible TIL growth. In addition, one or more aspects (e.g., but notlimited to, culturing and/or expanding) of the inventive methods may beautomatable. The development of a simpler, less expensive, and lesslabor-intensive method to generate clinically effective TIL is believedto advantageously aid in the more widespread use of adoptive celltherapy and permit the delivery of therapeutically effective TIL to morepatients in a shorter time period. Faster and more efficient adoptivecell therapy may allow patients to be treated more quickly when thedisease is at an earlier, less progressive stage, which increases thelikelihood that more patients will respond positively to treatment. Theinventive methods may also make it possible to treat certain patientswho previously may not have been successfully treated because sufficientnumbers of TIL were not generated due to the technical and logisticalcomplexities of methods that do not use gas permeable flasks.Accordingly, the inventive methods advantageously may make it possibleto treat or prevent a wider variety of cancers and, therefore, treat alarger number of patients.

The method comprises obtaining a tumor tissue sample from the mammal.The tumor tissue sample can be obtained from numerous sources, includingbut not limited to tumor biopsy or necropsy. The tumor tissue sample maybe obtained from any cancer, including but not limited to any of thecancers described herein. Preferably, the cancer is melanoma. The tumortissue sample may be obtained from any mammal. Preferably, the tumortissue sample is obtained from a human. In an embodiment, the tumortissue sample may be a tumor tissue fragment. The tumor tissue samplemay be fragmented, e.g., by dissection, to provide a tumor tissuefragment. Alternatively or additionally, the tumor tissue sample may,optionally, be enzymatically or mechanically digested. Suitable enzymesfor fragmenting the tumor tissue sample include, but are not limited to,collagenase. In an embodiment, the tumor tissue sample is fragmentedwithout digestion. The tumor tissue fragment may be any suitable size.Preferably, the tumor tissue fragment has a size of about 1 mm³ or lessto about 8 mm³ or larger, about 1 mm³ to about 4 mm³, about 1 mm³ toabout 2 mm³, or about 1 mm³.

The method further comprises culturing the tumor tissue sample in afirst gas permeable container containing cell medium therein. In anembodiment, the tumor tissue sample is cultured directly on the gaspermeable material in the gas permeable container without digestion. Inanother embodiment, an enzymatically or mechanically digested tumortissue sample may be cultured directly on the gas permeable material.Any suitable cell medium may be used. The cell culture medium mayfurther comprise any suitable T-cell growth factor such as, e.g.,interleukin (IL)-2. The cell culture medium may optionally furthercomprise human AB serum. The tumor tissue sample may contain TIL thatare autologous to the patient. Culturing the tumor tissue sample mayinclude culturing the TIL present in the tumor sample.

The method also comprises obtaining TIL from the tumor tissue sample.The tumor tissue sample comprises TIL. As the tumor tissue sample iscultured in the gas permeable container, e.g., on gas permeable materialin the container, TIL present in the tumor tissue sample also begin togrow in the gas permeable container, e.g., on the gas permeablematerial. TIL may be obtained from the tumor tissue sample in anysuitable manner.

The first gas permeable container may be any suitable gas permeablecontainer. In an embodiment of the invention, the first gas permeablecontainer comprises a base, sides, and a cap. The container, preferablythe base, may comprise a gas permeable support and a gas permeablematerial, e.g., a gas permeable membrane. The gas permeable material maybe positioned inside the container directly on the gas permeable supportwhich comprises openings (e.g., channels) in fluid communication withambient gas in order to facilitate gas exchange between the interior ofthe container and the ambient gas. The cap may comprise a vent and/or aport (e.g., an access port). In a preferred embodiment, the access portmay have an opening greater than about 1 mm to about 1 cm (e.g., greaterthan about 1 mm or greater than about 1 cm). An access port with anopening greater than about 1 mm to about 1 cm may advantageouslyeliminate or reduce disturbance of the TIL. In an embodiment, the gaspermeable container may comprise a vent or a vented port, which may beadvantageous in the event that the temperature in the container dropsduring handling. Preferably the first gas permeable container is a gaspermeable container as described in U.S. Patent Application PublicationNo. 2005/0106717, which is incorporated herein by reference, andcommercially available from Wilson Wolf Manufacturing Corporation (e.g.,G-Rex10, GP200, G-Rex100, GP2000 containers) (New Brighton, Minn.).

The first gas permeable container may have any suitable cell mediumvolume capacity. For example, the first gas permeable container may havea medium volume capacity of about 40 mL or more; about 200 mL or more;about 500 mL or more; about 2,000 mL or more; or about 5,000 mL or more.Although the first gas permeable container may have any suitable mediumvolume capacity, the tumor tissue sample and/or TIL may be cultured inany suitable volume of medium. Preferably, the tumor tissue sampleand/or TIL are cultured submerged under a height of at least about 1.3cm of cell culture medium. More preferably, the tumor tissue sampleand/or TIL are cultured submerged under a height of at least about 2.0cm of cell culture medium. Tumor tissue samples and/or TIL cultured on agas permeable material submerged under a height of at least about 1.3 cmor a height of at least about 2.0 cm of medium may, advantageously, behandled and fed less frequently.

In addition, the first gas permeable container may provide any suitablesurface area for the growth of the TIL. For example, the gas permeablecontainer may have a surface area for growth of the TIL of about 10 cm²or more; about 100 cm² or more; or about 650 cm² or more.

In use, the tumor tissue sample and/or TIL are cultured inside the firstgas permeable container in contact with the gas permeable material andsubmerged under a suitable volume of culture medium. Culturing the tumortissue sample and/or TIL in contact with the gas permeable materialfacilitates gas exchange between the cells and the ambient air.Facilitating gas exchange between the cells and the ambient airfacilitates the respiration, growth, and viability of the cells.Moreover, the gas exchange across the gas permeable material canfacilitate circulation of the medium (e.g., by convection and diffusion)within the container, which facilitates feeding of the TIL.

The method further comprises expanding the number of TIL in a second gaspermeable container containing cell medium therein using irradiatedallogeneic feeder cells and/or irradiated autologous feeder cells. In anembodiment, the number of TIL is expanded using a ratio of about 1 TILto at least about 20 feeder cells, about 1 TIL to at least about 25feeder cells, about 1 TIL to at least about 50 feeder cells, about 1 TILto at least about 100 feeder cells, about 1 TIL to at least about 200feeder cells, e.g., a TIL-to-feeder cell ratio of about 1 to about 20,about 1 to about 25, about 1 to about 50, about 1 to about 100, or about1 to about 200. The second gas permeable container may be as describedfor the first container.

The cultured TIL are expanded, preferably, rapidly expanded. Rapidexpansion provides an increase in the number of TIL of at least about50-fold (or 60-, 70-, 80-, 90-, or 100-fold, or greater) over a periodof about 10 to about 14 days, preferably about 14 days. More preferably,rapid expansion provides an increase of at least about 200-fold (or300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period ofabout 10 to about 14 days, preferably about 14 days. Most preferably,rapid expansion provides an increase of at least about 1000-fold over aperiod of about 10 to about 14 days, preferably about 14 days.Preferably, rapid expansion provides an increase of about 1000-fold toabout 2000-fold, e.g., about 1000-fold, about 1500-fold, or about2.000-fold over a period of about 14 days.

Expansion can be accomplished in the gas permeable container by anysuitable method. For example, TIL can be rapidly expanded usingnon-specific T-cell receptor stimulation in the presence of feeder cells(e.g., irradiated allogeneic feeder cells, irradiated autologous feedercells, and/or artificial antigen presenting cells (e.g., K562 leukemiacells transduced with nucleic acids encoding CD3 and/or CD8)) and eitherinterleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 beingpreferred. In an embodiment of the method, expanding the number of TILuses about 1×10⁹ to about 4×10⁹ allogeneic feeder cells and/orautologous feeder cells, preferably about 2×10⁹ to about 3×10⁹allogeneic feeder cells and/or autologous feeder cells. The non-specificT-cell receptor stimulus can include, for example, about 30 ng/ml ofOKT3, a mouse monoclonal anti-CD3 antibody (available from ORTHO-MCNEIL,Raritan, N.J. or MILTENYI BIOTECH, Auburn, Calif.). Alternatively, TILcan be rapidly expanded by, for example, stimulation of the TIL in vitrowith an antigen (one or more, including antigenic portions thereof, suchas epitope(s), or a cell) of the cancer, which can be optionallyexpressed from a vector, such as an human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27L) or gp100:209-217(210M), in the presence of a T-cell growth factor, such as 300 IU/mlIL-2 or IL-15, with IL-2 being preferred. Other suitable antigens mayinclude, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen,MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. The invitro-induced TIL are rapidly expanded by re-stimulation with the sameantigen(s) of the cancer pulsed onto HLA-A2-expressingantigen-presenting cells. Alternatively, the TIL can be re-stimulatedwith, for example, irradiated, autologous lymphocytes or with irradiatedHLA-A2+ allogeneic lymphocytes and IL-2, for example.

In an embodiment, expanding the number of TIL may comprise using about5,000 mL to about 10,000 mL of cell medium, preferably about 5,800 mL toabout 8,700 mL of cell medium. In an embodiment, expanding the number ofTIL uses no more than one type of cell culture medium. Any suitable cellculture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50μg/ml streptomycin sulfate, and 10 μg/ml gentamicin sulfate) cellculture medium (Invitrogen, Carlsbad Calif.). In this regard, theinventive methods advantageously reduce the amount of medium and thenumber of types of medium required to expand the number of TIL.

In an embodiment, expanding the number of TIL may comprise feeding thecells no more frequently than every third or fourth day. Expanding thenumber of cells in a gas permeable container advantageously simplifiesthe procedures necessary to expand the number of cells by reducing thefeeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. Without being bound to a particulartheory, it is believed that particulate serum components present in somecell medium supplements (e.g., AB serum) have little or no detrimentaleffects on TIL growth. The use of unfiltered cell medium may,advantageously, simplify the procedures necessary to expand the numberof cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container lacks beta-mercaptoethanol (BME). The absence of BMEfrom the cell medium may be advantageously more compliant with cGMP and,thus, may advantageously make it easier to gain regulatory approval.

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TIL from the tumor tissue sample; expanding the number of TILin a second gas permeable container containing cell medium therein usingirradiated allogeneic feeder cells and/or irradiated autologous feedercells may be about 28 to about 42 days, e.g., about 28 days.

The method comprises administering the expanded TIL to the mammal. TheTIL can be administered by any suitable route as known in the art.Preferably, the TIL are administered as an intra-arterial or intravenousinfusion, which preferably lasts about 30 to about 60 minutes. Otherexamples of routes of administration include intraperitoneal,intrathecal and intralymphatic.

Likewise, any suitable dose of TIL can be administered. Preferably, fromabout 1.0×10¹⁰ TIL to about 13.7×10¹⁰ TIL are administered, with anaverage of around 5.0×10¹⁰ TIL, particularly if the cancer is melanoma.Alternatively, from about 1.2×10¹⁰ to about 4.3×10¹⁰ TIL areadministered.

In addition to TIL, macrophages, monocytes, and natural killer (NK)cells may also be obtained from the tumor tissue sample, cultured, andexpanded as described herein for TIL. Accordingly, the method may alsocomprise administering macrophages, monocytes, and natural killer (NK)cells to the mammal. The inventive methods may also be effective forexpanding NK cells.

In an embodiment of the method, a T-cell growth factor that promotes thegrowth and activation of the TIL is administered to the mammal eitherconcomitantly with the TIL or subsequently to the TIL. The T-cell growthfactor can be any suitable growth factor that promotes the growth andactivation of the TIL. Examples of suitable T-cell growth factorsinclude interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be usedalone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15,IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15,or IL-12 and IL-2. IL-2 is a preferred T-cell growth factor.

In an embodiment of the method, the TIL are modified to express a T-cellgrowth factor that promotes the growth and activation of the TIL.Suitable T-cell growth factors include, for example, any of thosedescribed above. Suitable methods of modification are known in the art.See, for instance, Sambrook et al., Molecular Cloning: A LaboratoryManual, 3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.2001; and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994. Desirably,modified TIL express the T-cell growth factor at high levels. T-cellgrowth factor coding sequences, such as that of IL-12, are readilyavailable in the art, as are promoters, the operable linkage of which toa T-cell growth factor coding sequence promote high-level expression. Inan embodiment, the TIL may be modified to express IL-12 as described inWorld Intellectual Property Organization Patent Application PublicationNo. WO 2010/126766, which is incorporated herein by reference.

In some embodiments, it is believed, two cytokines are more effectivethan a single cytokine, and three cytokines, e.g., IL-2, IL-7 and IL-15,are more effective than any two cytokines. It is believed that IL-15enhances a tumor-specific CD8⁺ T-cell response. In this regard, theadministration of IL-15-cultured cells with IL-2 (such as a bolusinjection) can be particularly efficacious. In another embodiment, TILmodified to express IL-12 may be administered with IL-2 as a bolusinjection.

The T-cell growth factor can be administered by any suitable route. Ifmore than one T-cell growth factor is administered, they can beadministered simultaneously or sequentially, in any order, and by thesame route or different routes. Preferably, the T-cell growth factor,such as IL-2, is administered intravenously as a bolus injection.Desirably, the dosage of the T-cell growth factor, such as IL-2, is whatis considered by those of ordinary skill in the art to be high.Preferably, a dose of about 720,000 IU/kg of IL-2 is administered threetimes daily until tolerance, particularly when the cancer is melanoma.Preferably, about 5 to about 15 doses of IL-2 are administered, with anaverage of around 8 doses.

TIL can recognize any of the unique antigens produced as a result of theestimated 10,000 genetic mutations encoded by each tumor cell genome.The antigen, however, need not be unique. TIL can recognize one or moreantigens of a cancer, including an antigenic portion of one or moreantigens, such as an epitope, or a cell of the cancer. An “antigen of acancer” and an “antigen of the cancer” are intended to encompass all ofthe aforementioned antigens. If the cancer is melanoma, such asmetastatic melanoma, preferably the TIL recognize MART-1 (such asMART-1:26-35 (27L)), gp100 (such as gp100:209-217 (210M)), or a “unique”or patient-specific antigen derived from a tumor-encoded mutation. Othersuitable melanoma antigens which may be recognized by TIL can include,but are not limited to, tyrosinase, tyrosinase related protein (TRP)1,TRP2, and MAGE. TIL can also recognize antigens such as, for example,NY-ESO-1, telomerase, p53, HER2/neu, carcinoembryonic antigen, orprostate-specific antigen, for treatment of lung carcinoma, breastcancer, colon cancer, prostate cancer, and the like.

In an embodiment of the method, the TIL are modified to express a T cellreceptor (TCR) having antigenic specificity for a cancer antigen, e.g.,any of the cancer antigens described herein. Suitable TCRs include, forexample, those with antigenic specificity for a melanoma antigen, e.g.,gp100 or MART-1. Suitable methods of modification are known in the art.See, for instance, Sambrook and Ausubel, supra. For example, the TIL maybe transduced to express a T cell receptor (TCR) having antigenicspecificity for a cancer antigen using transduction techniques describedin Morgan et al., Science 314(5796):126-9 (2006) and Johnson et al.Blood 114:535-46 (2009).

The cancer can be any cancer, including any of acute lymphocytic cancer,acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, braincancer, breast cancer, cancer of the anus, anal canal, or anorectum,cancer of the eye, cancer of the intrahepatic bile duct, cancer of thejoints, cancer of the neck, gallbladder, or pleura, cancer of the nose,nasal cavity, or middle ear, cancer of the oral cavity, cancer of thevulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesenterycancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer,skin cancer, small intestine cancer, soft tissue cancer, stomach cancer,testicular cancer, thyroid cancer, ureter cancer, and urinary bladdercancer. A preferred cancer is melanoma. A particularly preferred canceris metastatic melanoma.

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

Promoting regression of cancer in a mammal may comprise treating orpreventing cancer in the mammal. The terms “treat,” “prevent,” and“regression,” as well as words stemming therefrom, as used herein, doesnot necessarily imply 100% or complete regression. Rather, there arevarying degrees of treatment, prevention, and regression of which one ofordinary skill in the art recognizes as having a potential benefit ortherapeutic effect. In this respect, the inventive methods can provideany amount of any level of treatment, prevention, or regression ofcancer in a mammal. Furthermore, the treatment, prevention, orregression provided by the inventive method can include treatment,prevention, or regression of one or more conditions or symptoms of thedisease, e.g., cancer. Also, for purposes herein, “treatment,”“prevention,” and “regression” can encompass delaying the onset of thedisease, or a symptom or condition thereof.

Another embodiment provides a method of obtaining an expanded number ofTIL from a mammal for adoptive cell immunotherapy comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TIL from the tumor tissue sample; expanding the number of TILin a second gas permeable container containing cell medium therein usingirradiated allogeneic feeder cells and/or irradiated autologous feedercells.

The method comprises obtaining a tumor tissue sample from the mammal.The tumor tissue sample may be obtained as described herein with respectto any embodiments of the invention.

The method comprises culturing the tumor tissue sample in a first gaspermeable container containing cell medium therein. The tumor tissuesample may be cultured in a first gas permeable container as describedherein with respect to any embodiments of the invention.

The method comprises obtaining TIL from the tumor tissue sample. The TILmay be obtained from the tumor tissue sample as described herein withrespect to any embodiments of the invention.

The method comprises expanding the number of TIL in a second gaspermeable container containing cell medium therein using irradiatedallogeneic feeder cells and/or irradiated autologous feeder cells. Thenumber of TIL may be expanded as described herein with respect to anyembodiments of the invention.

Still another embodiment of the invention provides a method of obtainingan expanded number of TIL from a mammal for adoptive cell immunotherapycomprising obtaining a tumor tissue sample from the mammal; obtainingTIL from the tumor tissue sample; expanding the number of TIL in a gaspermeable container containing cell medium therein using irradiatedallogeneic feeder cells and/or irradiated autologous feeder cells.Obtaining a tumor tissue sample from the mammal, obtaining TIL from thetumor tissue sample, and expanding the number of TIL in a second gaspermeable container containing cell medium therein using irradiatedallogeneic feeder cells and/or irradiated autologous feeder cells may becarried out as described herein with respect to any embodiments of theinvention.

The method may further comprise culturing the tumor tissue by anysuitable method that facilitates the obtaining of TIL from the tumortissue sample. In this regard, culturing the tumor tissue may compriseestablishing multiple independent cultures, e.g., microcultures. Forexample, culturing the tumor tissue may comprise culturing tumorfragments in plates, e.g., 24-well plates. In an embodiment, the tumortissue is cultured without a gas permeable container.

In some embodiments, the method further comprises selecting TIL capableof lysing cancer cells while in other embodiments, the method does notinclude selecting TIL capable of lysing cancer cells. TIL capable oflysing cancer cells may be selected by identifying TILs having anysuitable trait associated with the lysis of cancer cells and/or theregression of cancer. Exemplary suitable TIL traits that may serve asthe basis for selecting TILs may include any one or more of IFN-γrelease upon co-culture with autologous tumor cells; cell surfaceexpression of one or more of CD8, CD27, and CD28; and telomere length.Without being bound to a particular theory, it is believed that cellsurface expression of one or more of CD8, CD27, and CD28 and longertelomere lengths are associated with positive objective clinicalresponses in patients and persistence of the cells in vivo. Preferablythe trait is IFN-γ release upon co-culture with autologous tumor cells.In an embodiment of the invention, selected TIL release about 200 pg/mlor more of IFN-γ upon co-culture with tumor cells.

In some embodiments, selecting TIL capable of lysing cancer cellscomprises testing individual cultures for presence of the trait andidentifying TIL possessing the trait. Methods of testing cultures forthe presence of any one or more of IFN-γ release upon co-culture withautologous tumor cells; cell surface expression of one or more of CD8,CD27, and CD28; and telomere length (longer telomeres being associatedwith regression of cancer) are known in the art.

Any number of cultures may be selected. For example, one, two, three,four, five, or more cultures may be selected. In embodiments in whichtwo or more cultures are selected, the selected cultures may be combinedand the number of TIL expanded in one (or more) gas permeablecontainers. Preferably, however, in embodiments in which two or morecultures are selected, each selected culture is separately expanded inseparate gas permeable containers. Without being bound to a particulartheory, it is believed that expanding multiple selected culturesseparately advantageously increases lymphocyte diversity for patienttreatment.

The method may further comprise expanding the number of TIL in anidentified culture in a second gas permeable container containing cellmedium therein using irradiated allogeneic feeder cells and/orirradiated autologous feeder cells as described herein with respect toany embodiments of the invention.

Another embodiment of the invention provides a method of promotingregression of cancer in a mammal comprising obtaining a tumor tissuesample from the mammal; obtaining TIL from the tumor tissue sample;expanding the number of TIL in a second gas permeable containercontaining cell medium therein using irradiated allogeneic feeder cellsand/or irradiated autologous feeder cells; and administering theexpanded number of TIL to the mammal. Obtaining a tumor tissue samplefrom the mammal, obtaining TIL from the tumor tissue sample, expandingthe number of TIL in a gas permeable container containing cell mediumtherein using irradiated allogeneic feeder cells and/or irradiatedautologous feeder cells, and administering the expanded number of TIL tothe mammal may be carried out as described herein with respect to anyembodiments of the invention. In some embodiments, the method furthercomprises selecting TIL capable of lysing cancer cells. The TIL may beselected as described herein with respect to any embodiments of theinvention.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES Initial TIL Culture

Patients were entered into clinical protocols and signed informedconsents that were approved by the Institutional Review Board of theNational Cancer Institute prior to tumor resection. TIL were initiallycultured from enzymatic tumor digests and tumor fragments (about 1 toabout 8 mm³) produced by sharp dissection. Tumor digests were generatedby incubation in enzyme media (Roswell Park Memorial Institute (RPMI)1640, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and1.0 mg/mL of collagenase) followed by mechanical dissociation(GentleMACS™ dissociator, Miltenyi Biotec, Auburn, Calif.). In brief,immediately after placing the tumor in enzyme media the tumor wasmechanically dissociated for approximately 1 minute. The solution wasthen incubated for 30 minutes at 37° C. in 5% CO₂ and was thenmechanically disrupted again approximately 1 minute. After beingincubated again for 30 minutes at 37° C. in 5% CO₂, the tumor wasmechanically disrupted a third time for approximately one minute. Ifafter the third mechanical disruption, large pieces of tissue werepresent, one or two additional mechanical dissociations were applied tothe sample, with or without 30 additional minutes of incubation at 37°C. in 5% CO₂. At the end of the final incubation, if the cell suspensioncontained a large number of red blood cells or dead cells, a densitygradient separation using FICOLL branched hydrophilic polysaccharide (GEHealthcare, Smyrna, Ga.) was preformed to remove these cells.

TIL growth from digests and fragments were initiated in either gaspermeable flasks with a 40 mL volume and a 10 cm² gas-permeable siliconbottom (G-Rex10, Wilson Wolf Manufacturing, New Brighton, Minn., USA) or24-well plates (Corning Corning, N.Y.). When TIL cultures were initiatedin 24-well plates (COSTAR 24-well cell culture cluster, flat bottom,Corning Incorporated, Corning, N.Y.), each well was seeded with 1×10⁶tumor digest cells or one tumor fragment approximately 1 to 8 mm³ insize in 2 mL of CM with IL-2 (6000 IU/mL, Chiron Corp., Emeryville,Calif.). CM included RPMI 1640 with glutamine, supplemented with 10% ABserum, 25 mM Hepes and 10 μg/ml gentamicin. When cultures were initiatedin G-Rex10 flasks, each flask was loaded with 10 to 40×10⁶ viable tumordigest cells or 5 to 30 tumor fragments in 10 to 40 ml of CM with IL-2.Both the G-Rex10 and 24-well plates were incubated in a humidifiedincubator at 37° C. in 5% CO₂ and five days after culture initiation,half the media was removed and replaced with fresh CM and IL-2 and afterday 5, half the media was changed every 2 to 3 days.

TIL Rapid Expansion Protocol (REP)

REP of TIL was performed using T-175 flasks and gas permeable bags aspreviously described (Tran et al., J. Immunother. 31(8):742-51 (2008);Dudley et al., J. Immunother. 26(4):332-42 (2003)) or gas permeablecultureware (G-Rex flasks). For TIL REP in T-175 flasks, 1×10⁶ TILsuspended in 150 ml of media was added to each T-175 flask. The TIL werecultured with irradiated (50 Gy) allogeneic peripheral blood mononuclearcells (PBMC) as “feeder” cells at a ratio of 1 TIL to 100 feeder cellsand the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium,supplemented with 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3.The T-175 flasks were incubated at 37° C. in 5% CO₂. Half the media wasexchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. Onday 7 cells from two T-175 flasks were combined in a 3 liter bag and 300mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was addedto the 300 ml of TIL suspension. The number of cells in each bag wascounted every day or two and fresh media was added to keep the cellcount between 0.5 and 2.0×10⁶ cells/mL.

For TIL REP in 500 mL capacity gas permeable flasks with 100 cm²gas-permeable silicon bottoms (G-Rex100, commercially available fromWilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×10⁶or 10×10⁶ TIL were cultured with irradiated allogeneic PBMC at a ratioof 1 to 100 in 400 mL of 50/50 medium, supplemented with 5% human ABserum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The G-Rex100flasks were incubated at 37° C. in 5% CO₂. On day 5,250 mL ofsupernatant was removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491×g) for 10 minutes. The TIL pellets werere-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IUper mL of IL-2, and added back to the original G-Rex100 flasks. When TILwere expanded serially in G-Rex100 flasks, on day 7 the TIL in eachG-Rex100 were suspended in the 300 mL of media present in each flask andthe cell suspension was divided into 3 100 mL aliquots that were used toseed 3 G-Rex100 flasks. Then 150 mL of AIM-V with 5% human AB serum and3000 IU per mL of IL-2 was added to each flask. The G-Rex100 flasks wereincubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000IU per mL of IL-2 was added to each G-Rex100 flask. The cells wereharvested on day 14 of culture.

Cell Counts, Viability, Flow Cytometery

The expression of CD3, CD4, CD8 and CD56 was measured by flow cytometrywith antibodies from BD Biosciences (BD Biosciences, San Jose, Calif.)using a FACSCanto™ flow cytometer (BD Biosciences). The cells werecounted manually using a disposable hemacytometer and viability wasassessed using trypan blue staining.

Cyokine Release Assays

TIL were evaluated for interferon-gamma (IFN-γ) secretion in response tostimulation either with OKT3 antibody or co-culture with autologoustumor digest. For OKT3 stimulation, TIL were washed extensively, andduplicate wells were prepared with 1×10⁵ cells in 0.2 ml CM in 96 wellflat-bottom plates pre-coated with 0.1 or 1.0 μg/mL of OKT-3 antibodydiluted in PBS. After overnight incubation, the supernatants wereharvested and IFN-γ in the supernatant was measured by ELISA(Pierce/Endogen, Woburn, Mass.). For the co-culture assay, TIL cellswere placed into a 96-well plate with autologous tumor cells. After a 24hour incubation, supernatants were harvested and IFN-γ release wasquantified by ELISA.

Statistical Analysis

Values are mean one± standard error of the mean (SEM) unless otherwiseindicated. Groups were compared using paired T tests.

Example 1

This example demonstrates that TIL cultured in gas-permeable containersis better than, or at least comparable to, that in a 24-well plate.

The growth of TIL from tumors using gas permeable flasks with a 40 mLcapacity and 10 cm² gas permeable silicone bottom (G-Rex10, Wilson WolfManufacturing Corporation, New Brighton, Minn., USA (providing about 10cm² of surface area for growth of the TIL)) or 24-well plates (CorningCorning, N.Y.) was compared. A total of 14 melanoma samples were tested,including 9 freshly prepared tumor digests (Table 1A) and 5 thawedsamples from previously frozen tumor digests (Table 1B). TIL from frozentumor from patient 2653 were not able to be cultured in either theG-Rex10 or 24-well plates. Except for one fresh sample (#3522), theratio of harvested TIL to initially seeded cells at day 17 to 29 wassimilar to or better in the G-Rex10 flasks than in the 24-well plate(Table 1A and 1B).

TABLE 1A Comparison of initial TIL culture in G-Rex10flasks with 24-wellplates using fresh tumor digests Ratio of # TIL harvested/# cell TILPhenotype (% seeded TIL Viability (%) expressing CD3+CD8+) Sample24-well 24-well 24-well Type Patient # plate G-Rex10 plate G-Rex10 plateG-Rex10 Fresh 3520 1.28 1.17 92.80 93.30 26.50 31.60 Tumor 3522 2.141.46 92.10 95.10 39.80 48.70 Digest 3523 2.34 2.30 94.50 93.60 15.0037.60 3524 1.27 6.22 80.20 95.00 76.70 86.60 3546 5.86 7.60 90.40 93.8044.60 37.30 3552 3.20 5.83 94.90 97.10 43.10 72.40 3556 3.60 4.15 96.2096.20 35.40 31.20 3560 6.06 6.25 95.80 97.20 32.30 38.60 3561 4.76 6.3894.70 93.90 65.80 83.40 Average ± SEM 3.39 ± 0.61 4.60 ± 0.80 92.40 ±1.65 95.02 ± 0.50 42.13 ± 6.33 51.93 ± 7.51

TABLE 1B Comparison of initial TIL culture in G-Rex10flasks with 24-wellplates using frozen tumor digests Ratio of # TIL harvested/# cell TILPhenotype (% seeded TIL Viability (%) expressing CD3+CD8+) Sample24-well 24-well 24-well Type Patient # plate G-Rex10 plate G-Rex10 plateG-Rex10 Frozen 2653 0.16 0.19 87.70 90.60 N.T. N.T. Tumor 3289 2.43 5.3095.10 98.00 70.40 73.60 Digest 2976 7.26 7.50 99.00 99.00 66.30 70.003071 1.79 6.05 88.40 97.70 37.50 29.10 2998 3.36 5.00 96.30 97.00 64.4075.80 Average ± SEM 3.00 ± 1.18 4.81 ± 1.23 93.30 ± 2.24 96.46 ± 1.5059.65 ± 6.70 62.12 ± 9.91 N.T. = Not tested.

The viability and percentage of cells expressing CD3 and CD8 betweenthese two types of vessels were similar (Tables 1A and 1B). TIL wereobtained from 13 of the 14 samples. These results suggest that TILgrowth in G-Rex10 is better than, or at least comparable to, that in a24-well plate.

IFN-γ production by TIL cultured in G-Rex10 flasks was also comparedwith that of TIL cultured in 24-well plates. IFN-γ production followingstimulation with autologous tumor by TIL from 4 patients cultured inboth types of vessels was similar (Table 1C)

TABLE 1C Interferon-γ release (pg/ml) by unstimulated andtumor-stimulated TIL Tumor Target¹ Patient Growth Method None AllogeneicAutologous 3552 plate 114 157 >1541 G-Rex10 81 338 >1577 3556 plate 90184 139 G-Rex10 87 363 282 3560 plate 135 175 318 G-Rex10 104 212 5723561 plate 70 97 207 G-Rex10 84 128 253 ¹Cryopreserved enzymaticallydigested single cell tumor suspension was thawed and 1 × 10⁵ viabletumor cells were cocultured with TIL (1:1 ratio) overnight beforequantifying interferon-γ in the supernatant by ELISA. Values in bold aremore than two times background and >200 pg/ml.

Example 2

This example demonstrates that the culture of TIL from tumor fragmentsin gas permeable flasks produces a greater number of TIL as compared toculture in 24-well plates after 7 to 13 days.

The growth of TIL from tumor fragments in G-Rex10 flasks or 24-wellplates was next compared. For each tumor sample, fragments approximately1 to 8 mm³ in size were seeded into 24-well plates at 1 piece per welland into G-Rex10 flasks at 5, 10, 20, or 30 pieces per flask. The growthof TIL from 2 lymph nodes and 1 liver metastasis was assessed (Table 2).TIL could be grown from tumor fragments in both gas-permeable flasks and24-well plates, but after 7 to 13 days greater quantities of TIL wereobtained from the G-Rex10 flasks than the wells (Table 2).

TABLE 2 Initial TIL culture using tumor fragments in G-Rex10 flasks and24-well plates. Patient Number of Cells Number and # of TIL TIL TILPhenotype Tumor Culture Tumor TIL# per Viability (% TIL expressing eachantigen) Source Vessel Fragments* (Day 7-13) fragment (%) CD3+ CD3+CD4+CD3+CD8+ CD56+ 3581 (Lymph Wells 1 per well, 5 total 2.71 × 10⁷ 5.42 ×10⁶ 93.2% 53.4% 40.2% 10.3% 11.6%  node) G- 5 fragments 4.29 × 10⁷ 8.58× 10⁶ 97.0% 61.0% 44.0% 14.3% 7.9% Rex10 G- 10 fragments 8.02 × 10⁷ 8.02× 10⁶ 95.4% 58.5% 42.2% 13.7% 9.3% Rex10 G- Dissociated tumor 4.82 × 10⁷2.41 × 10⁶ 96.4% 60.3% 40.1% 16.3% 8.9% Rex10 3584 (Liver) Wells 1 perwell, 5 total 1.52 × 10⁷ 3.04 × 10⁶ 92.7% 89.2% 19.8% 68.8% 5.3% G- 5fragments 4.38 × 10⁷ 8.76 × 10⁶ 95.7% 92.0% 10.6% 81.1% 2.1% Rex10 G- 10fragments 8.13 × 10⁸ 8.13 × 10⁶ 94.4% 91.4% 9.8% 81.2% 2.3% Rex10 G-Dissociated tumor 4.94 × 10⁷ 2.60 × 10⁶ 95.8% 87.7% 11.7% 75.6% 3.3%Rex10 3585 (Lymph Wells 1 per well, 5 total 2.34 × 10⁷ 4.68 × 10⁶ 95.8%85.5% 14.4% 68.6% 4.20%  node) G- 5 fragments 6.41 × 10⁷ 12.8 × 10⁶95.0% 92.1% 20.6% 70.7% 1.10%  Rex10 G- 10 fragments 1.12 × 10⁸ 11.2 ×10⁶ 97.2% 93.4% 18.6% 73.9% 1.30%  Rex10 G- Dissociated tumor 3.33 × 10⁷1.23 × 10⁶ 90.0% 90.4% 24.0% 65.7% 2.60%  Rex10 *Each tumor fragment wasapproximately 1 mm³.

A total of 11 tumor samples collected from 9 patients were tested; 3samples were from 1 patient, but they were from different metastatictumors. TIL could be grown from tumor fragments from all 11 samples inboth the G-Rex10 flasks and 24-well plates, but after 7 to 23 days inculture greater quantities of TIL were obtained from the G-Rex10 flasks.The head-to-head comparison of culturing 10 fragments in the two typesof vessels showed that TIL yields from G-Rex10 flasks were consistentlyhigher than those from 24-well plate (FIG. 1A). The optimal numbers offragments seeded into each G-Rex10 flasks was further assessed. Thequantities of TIL obtained per tumor fragment decreased as the number ofpieces added to each G-Rex10 flask increased (FIG. 1B), however, totalTIL yield was higher as more fragments were cultured in the G-Rex10flasks until 20 or more tumor fragments were cultured in each G-Rex10flask (FIG. 1C). The viability of TIL obtained from G-Rex10 flasks wassimilar to that of TIL obtained from 24-well plates (96.6±0.6% vs95.3±0.8%) as was the proportion of TIL expressing CD3 and CD8(67.8±7.2% vs 63.3±7.7%). TIL were also obtained from 3 of the 11samples by the culture of mechanically dissociated samples in G-Rex10flasks, but greater yields were obtained using tumor fragments as thestarting material.

Example 3

This example demonstrates the kinetics of TIL growth in gas-permeableflasks.

In order to assess the kinetics of TIL growth in gas-permeablecultureware, TIL from one patient were cultured in G-Rex100 flasksseeded at a density of 5×10⁶ and 10×10⁶ cells per flask. The cells werecounted daily after day 6. On Day 6 the number of cells in the G-Rex100flask seeded at 5×10⁶ cells was 255×10⁶ cells and at 10×10⁶ cells was300×10⁶ cells. The quantity of TIL in G-Rex100 flasks seeded at eachcell density increased steadily until day 9, but there was littleincrease in cell counts between days 9 and 10. After 10 days 906×10⁶cells were harvested from flasks seeded with 5×10⁶ TIL and 1,050×10⁶cells from flasks seeded with 10×10⁶ TIL. Although TIL expanded well for9 days, in order to keep the G-Rex100 flask expansion process similar toREP in T-flasks and gas-permeable bags where TIL are transferred fromT-flasks to bags on day 7, further studies focused on TIL expansion inthe G-Rex100 flasks for 7 days.

Example 4

This example demonstrates that a 7-day culture of TIL in a gas permeablecontainer seeded with 10×10⁶ cells does not produce a significantlygreater number of cells than a gas permeable container seeded with 5×10⁶cells.

The first step in TIL rapid expansion protocol (REP) has traditionallybeen performed in T-175 flasks. The expansion of TIL in T-175 flasks wascompared to expansion in G-Rex100 flasks (providing about 100 cm² ofsurface area for growth of the TIL). The expansion of TIL from 4patients over 7 days in G-Rex100 flasks seeded with 5×10⁶ and 10×10⁶cells was compared with TIL expansion in T-175 flasks (Table 3). T-175flasks were seeded with 1×10⁶ cells.

TABLE 3 Comparison of TIL rapid expansion process (REP) over 7 days inT-175 flasks* and G-Rex100 flasks seeded with 5 × 10⁶ or 10 × 10⁶ TIL.Fold Increase Viability (%) Phenotype (% CD3+CD8+) G-Rex100 G-Rex100G-Rex100 G-Rex100 G-Rex100 G-Rex100 Patient T-175 5 × 10⁶ 10 × 10⁶ T-1755 × 10⁶ 10 × 10⁶ T-175 5 × 10⁶ 10 × 10⁶ 2812  74  88  57 95.8 96.6 94.766.4 64.6 60.5 3289 183 218 139 96.4 94.8 95.1 79.7 83.7 84.1 2976 254250 ND 94.1 96.1 ND 55.2 59.9 ND 3071 156 146 ND 87.4 90.3 ND 44.6 41.6ND Mean 167 ± 74 176 ± 73 98 ± 58 93.5 ± 2.9 94.5 ± 2.9 94.9 ± 0.3 61.5± 15.0 62.5 ± 17.3 72.3 ± 16.7 *T-175 flasks were seeded with 1 × 10⁶TIL.

After 7 days of culture of TIL from 4 patients, the number of cells inT-175 flasks increased to 206±103×10⁶ cells which represented anexpansion of 167±74 fold (Table 3). The culture of TIL from the same 4patients in G-Rex100 flask seeded with 5×10⁶ cells resulted in theproduction of 877±365×10⁶ cells which represented an expansion of 176±73fold. The culture of TIL from 2 of the 4 patients in G-Rex100 flaskseeded with 10×10⁶ cells resulted in the production of 980±580×10⁶ cellswhich represented an expansion of 98±58 fold. The viability andproportion of cells that expressed CD3 and CD8 were similar among thoseproduced by the three different conditions (Table 3). These resultssuggest that the performance of G-Rex100 flasks seeded at the lowerseeding density was comparable to that of the T-175 flasks. Since the7-day culture of TIL in G-Rex100 flasks seeded with 10×10⁶ cells did notproduce a significantly greater number of cells than the G-Rex 100 flaskseeded with 5×10⁶ cells, the lower seeding density was chosen for futureexperiments.

Example 5

This example demonstrates that a similar number of cells can be producedin a 500 mL gas permeable container as compared to a 2000 mL gaspermeable container.

Since the maximum cell yield from one G-Rex100 flask reached a plateauafter approximately 9 days, the production of adequate quantities of TILfor clinical therapy requires the splitting of cells during. REP andtransferring the cells into multiple gas-permeable flasks for furtherculture. TIL REP by serial culture was tested in G-Rex100L, another typeof gas-permeable flask that is commercially available for large scalecell expansion (Wilson Wolf Manufacturing Corporation, New Brighton,Minn., USA). The G-Rex100L has the same gas permeable surface area onthe silicone bottom of the flask as the G-Rex100 (providing about 100cm² of surface area for growth of the TIL), but the G-Rex100L is taller.As a result, the media capacity of the G-Rex100L flask is approximately2000 ml compared to approximately 500 mL for the G-Rex100.

TIL expansion was compared in these two types of flasks. TIL wereinitially seeded at a density of 5×10⁶ cells for both the G-Rex100 andG-Rex100L flasks, and were cultured for 7 days as described in Example3. After 7 days the cells from the G-Rex100 flask were split into 3equal parts, and seeded into 3 G-Rex100L flasks. The cells from theG-Rex100L flask were split into two equal parts, seeded into 2 G-Rex100Lflasks. The TIL were cultured for an additional 7 days in the G-Rex100and G-Rex100L flasks.

The expansion of TIL from two patients was compared in the G-Rex100 andG-Rex100L flasks. Both patients' TIL growth slowed after 13 or 14 days.The total number of cells produced after 14 days by culture in the 3G-Rex100 flasks and the 2 G-Rex100L flasks was similar for one patient(about 9×10⁹ cells in G-Rex100 and G-Rex100L after 14 days) but wasgreater in the G-Rex100 flask for the second (about 12×10⁹ cells inG-Rex100 and 8.4×10⁹ cells in G-Rex100L after 14 days)). Since a similarvolume of media is required to produce a similar number of cells in theG-Rex100 and in the G-Rex100L flasks and since the G-Rex100 flasks areeasier to handle, initial expansion of TIL in one G-Rex100 flaskfollowed by expansion in 3 G-Rex100 flasks was chosen for futureexperiments.

Example 6

This example demonstrates the consistency of serial TIL expansion usinggas permeable containers and a “full scale” expansion of TIL using gaspermeable containers.

The consistency of serial TIL expansion in G-Rex100 flasks using cellsfrom 14 patients was tested. Initially, 5×10⁶ TIL were seeded into aG-Rex100 flask and the cells were cultured for 7 days. They were thensplit into 3 equal parts, seeded into 3 G-Rex100 flasks. After 14 daysin culture, 8.60×10⁹±2.80×10⁹ TIL with a range of 2.24×10⁹ to 12.8×10⁹were produced. The number of TIL produced after 14 days was similar for12 patients, but lower for two others. When the 2 patients with thelowest overall TIL expansion were excluded, the mean quantity of TILproduced was 9.55×10⁹ cells per original G-Rex100 flask. The mean cellconcentration in G-Rex100 flasks at the end of the culture was 7.95×10⁶cells per mL.

The IFN-γ release from TIL produced by G-Rex100 REP and T-175 flask/bagREP was compared. TIL samples produced by both REP methods using thesame tumor samples from 4 patients were tested. Following stimulation byanti-CD3 IFN-γ production by TIL expanded in G-Rex100 flasks was similarto that of TIL expanded in T-175 flasks and bags (Table 4A).

TABLE 4A IFN-γ release (pg/ml)² Patient¹ Sample OKT3 1.0 μg/ml OKT3 1.0μg/ml None 3536 Flask/Bag 631, 672 457, 390 0, 0 G-Rex100 579, 553 243,277 0, 0 3539 Flask/Bag 12272, 14350 10553, 11039 179, 176 G-Rex10029792, 29550 26670, 23835 73, 80 3135 Flask/Bag  831, 1124 704, 643 0, 0G-Rex100 581, 635 151, 74  2, 1 3533 Flask/Bag 6870, 6370 4280, 500 114, 146 G-Rex100 5100, 4510 1513, 1407 136, 150 ¹TIL cells werestimulated by overnight incubation on plate bound OKT3 (anti-CD3antibody) coated at the concentration indicated. ²Values are IFN-γ(pg/ml) detected in duplicate wells measured by ELISA.

These results suggested that 20 to 30×10⁹ TIL could be produced by theinitial culture of 15×10⁶ TIL in 3 G-Rex100 flasks for 7 days followedby a second 7 day culture in 9 G-Rex100 flasks (Table 4B).

TABLE 4B Day 5 Day 11 Day 0 Medium Day 7 Media Day 14 Seeding ChangeSplit (1:3) Addition Harvest/Wash Containers 3 G-Rex100 3 G-Rex100 9G-Rex100 9 G-Rex100 Final 400 mL/ 300 mL 250 mL 400 mL Volume ofcontainer Medium Steps 5 × 10⁶ cells Remove 250 mL 300 mL for 250 mL inTIL are TIL are of each G- each flask harvested, seeded in 400 mL media;spin Rex100 is and 150 mL pooled and in each down and split into 3 newmedia washed of 3 G- resuspend 100 mL Rex100 TIL in 150 mL aliquots; 100mL flasks and add added to to remaining 3 G-Rex100 150 mL flasks; anadditional 150 mL of media is added

To test this “full scale” G-Rex100 REP, 15×10⁶ TIL from one patient weredivided among three G-Rex100 flasks, A, B and C. After 7 days in culturethe TIL in each flask were split into 3 equal parts, seeded into 3G-Rex100 flasks and cultured for an additional 7 days. The mean numberof TIL harvested from each of the 3 G-Rex100 flasks used for initialexpansion was 875×10⁶±30.8×10⁶ and ranged from 849×10⁶ to 909×10⁶ TILand the mean number harvested from each of the 9 G-Rex100 flasks usedfor the secondary expansion was 2.63±0.09×10⁹ and ranged for 2.55×10⁹ to2.70×10⁹ TIL (Table 5).

TABLE 5 Number of cells produced by a two-step G-Rex100 TIL rapidexpansion protocol (REP); the first step involved growth in 3 G-Rex100flasks and the second growth in 9 G-Rex100 flasks First G- Rex100 SecondG- Flask Day 0 Day 7 Rex100 Flask Day 11 Day 14 Post Wash A 5 × 10⁶ 849× 10⁶ A1 1.44 × 10⁹ 2.67 × 10⁹ A2 N.T. 2.43 × 10⁹ A3 N.T. 2.66 × 10⁹ B 5× 10⁶ 867 × 10⁶ B1 1.55 × 10⁹ 2.70 × 10⁹ B2 N.T. 2.66 × 10⁹ B3 N.T. 2.76× 10⁹ C 5 × 10⁶ 909 × 10⁶ C1 1.44 × 10⁹ 2.62 × 10⁹ C2 N.T. 2.60 × 10⁹ C3N.T. 2.55 × 10⁹ Total 15 × 10⁶  2.63 × 10⁹    14 × 10⁹ 23.6 × 10⁹ 21.0 ×10⁹

The total TIL yield was 23.6×10⁹ and 21.0×10⁹ remained after washing thecells. The viability of the cells was 96% and 69% of the cells expressedCD3 and CD8.

Cell potency, in terms of interferon (IFN)-γ secretion was also testedusing Enzyme-linked immunosorbent assay (ELISA). The cells grown usingthe gas-permeable G-Rex100 containers and the cells grown using non-gaspermeable containers produced a comparable amount of IFN-γ.

Example 7

This example demonstrates the rapid expansion of TIL using one 5000 mLgas permeable container.

While serial expansion of TIL in G-Rex100 flasks required far lessregents, less incubator space, less labor and less specialized equipmentthan REP in T-flasks and gas-permeable bags, it was hypothesized thatthe gas-permeable cultureware REP process could be further improved byusing one large vessel rather than 9 G-Rex100 flasks. Therefore, alarger vessel with a gas-permeable membrane with approximately 6.5 timesthe gas permeable surface area and 10 times the volume of the G-Rex100flasks was tested. This vessel was the GP5000 (providing about 650 cm²of surface area for growth of the TIL). Two different REP methods usingGP5000 gas-permeable vessels were tested. One method involved an initial7-day expansion in 2 G-Rex100 flasks, each seeded with 5×10⁶ TIL,followed by another expansion of the harvested TIL in a single GP5000vessel. The other method involved a single 14-day expansion of 10×10⁶TIL in a GP5000 vessel.

Cells from 2 patients were tested and the TIL yield of the two REPmethods were similar for both donors. Approximately 25×10⁹ TIL wereharvested from patient 3524 and approximately 20×10⁹ from patient 3560.The cell viability for all four REPs was >96% and >92% of patient 3524cells expressed CD8 and >35% of patient 3560 cells expressed CD8.

Example 8

This example demonstrates a clinical TIL production process.

Clinical scale TIL production using tumor fragments from 3 patients wasnext tested by initially culturing TIL in G-Rex10 flasks followed by REPin G-Rex100 flasks. For each patient, 6 G-Rex10 flasks were seeded with5 tumor fragments and after 14 to 15 days 5×10⁶ TIL from each G-Rex10flask were seeded into one G-Rex100 flask. After 7 days TIL from eachG-Rex100 flask were split into 3 G-Rex100 flasks and after an additional7 days in culture TIL were harvested from the 18 G-Rex100 flasks. Fortwo patients, 3613 and 3618, enough TIL could be harvested from each ofthe 6 G-Rex10 flasks for TIL REP in a G-Rex100 flask.

The quantity of TIL harvested from each of the G-Rex10 flasks rangedfrom 47.5 to 97.8×10⁶ cells for patient 3613 and 24.6 to 64.2×10⁶ forpatient 3618 (Table 6A). For patient 3625 sufficient quantities of TILwere obtained from 4 of the 6 G-Rex10 flasks. The quantity harvestedfrom these 4 flasks ranged from 59.7 to 140×10⁶ cells (Table 6A). Forpatients 3613 and 3618, 5×10⁶ TIL from each of the 6 G-Rex10 flasks wasseeded into a G-Rex100 flask. For patient 3625, 5×10⁶ TIL from 2 G-Rex10flasks were each seeded into one G-Rex100 flask and 10×10⁶ TIL from theother 2 G-Rex10 flasks were split and used to seed 4 G-Rex100 each with5×10⁶ TIL. At the completion of REP using patient 3613 cells 22.4×10⁹TIL were harvested, while REP using patient 3618 cells yielded 52.7×10⁹TIL and patient 3625 cells yielded 61.0×10⁹ TIL. The number of TILproduced by each of the 6 sets of 3 G-Rex100 flasks was similar for eachpatient. These results show that G-Rex100 flasks can produce sufficientquantities of TIL for clinical therapy using TIL initially cultured fromtumor fragments in G-Rex10 flasks. The same G-Rex100 REP protocol wasalso successful in expanding TIL that were initially cultured from tumorfragments in 24-well plates. There were no significant differences infold expansion using either TIL initially cultured from tumor fragmentsin 24-well plates or G-Rex10 flasks.

TABLE 6A TIL # of Phenotype Culture Fragments Day TIL # TIL Viability (%CD3+ Patient # Vessel Seeded Harvested Harvested #TIL/Fragment (%) CD8+)3613 1 5 14 47.5 × 10⁶ 9.50 × 10⁶ N.T. 70 2 5 14 94.5 × 10⁶ 18.9 × 10⁶N.T. 74 3 5 14 57.5 × 10⁶ 11.5 × 10⁶ N.T. 74 4 5 14 97.8 × 10⁶ 19.6 ×10⁶ N.T. 73 5 5 14 55.0 × 10⁶ 11.0 × 10⁶ N.T. 67 6 5 14 67.0 × 10⁶ 13.4× 10⁶ N.T. 67 3618 1 5 14 61.0 × 10⁶ 12.2 × 10⁶ 99.0 77 2 5 14 64.2 ×10⁶ 12.8 × 10⁷ 97.0 84 3 5 14 24.6 × 10⁶ 4.92 × 10⁶ 99.2 N.T. 4 5 1464.2 × 10⁶ 12.8 × 10⁶ 96.4 86 5 5 14 40.2 × 10⁶ 8.04 × 10⁶ 97.1 77 6 514 57.0 × 10⁶ 11.4 × 10⁶ 99.3 85 3625 1 5 15 86.0 × 10⁶ 17.2 × 10⁶ 96.873 2 5 15 59.4 × 10⁶ 11.9 × 10⁶ 98.7 91 3 5 15 71.8 × 10⁶ 14.4 × 10⁶98.6 73 4 5 15 1.80 × 10⁶** 0.36 × 10⁶ 64.3 N.T. 5 5 15 2.20 × 10⁶**0.440 × 10⁶  78.6 N.T. 6 5 15  140 × 10⁶ 28.0 × 10⁶ 100.0 74 N.T. = NotTested **Insufficient number of cells for clinical REP

Example 9

This example demonstrates that expanding TIL in gas permeable containersuses a lower number of containers, lower number of feeder cells, andlower amount of medium as compared to methods in which the TIL areexpanded in non-gas permeable containers.

TIL are expanded as described above using gas permeable containers(G-Rex100 and GP5000) and in non-gas permeable containers (bags and T175 flask). A comparison of the numbers of containers and amountsreagents used is set forth in Table 6B.

TABLE 6B No. of No. of No. of TIL Feeder Volume of Containers seededCells Medium T 175 Flask 20-40 Flasks 20 × 10⁶- 4 × 10⁹- ~30,000 ml 40 ×10⁶ 8 × 10⁹ (requires 2 different types of REP medium) LIFECELL 10-20Bags 20 × 10⁶- 4 × 10⁹- ~30,000 ml flasks (Baxter) 40 × 10⁶ 8 × 10⁹ 500mL gas 12-18 20 × 10⁶- 2 × 10⁹- 5800 ml-8700 ml permeable 30 × 10⁶ 2 ×10⁹ (only 1 REP container (G- medium is Rex100) necessary) 5000 mL gas 220 × 10⁶- 2 × 10⁹- 5800 ml-8700 ml permeable 130 × 0⁶ 2 × 10⁹ (only 1REP container medium is (GP5000) necessary)

Example 10

This example demonstrates that culturing TIL in unfiltered cell mediumhas little or no detrimental effects on TIL growth.

TIL (4×10⁶) were cultured in filtered or unfiltered complete medium (CM)(50 mL) (RPMI 1640 with glutamine, supplemented with 10% AB serum, 25 mMHepes and 10 μg/ml gentamicin) in four wells. On day 7/8, fold increase,viability, and % CD3+CD8+ were measured. The results are shown in Table7.

TABLE 7 Fold Increase Viability % CD3+CD8+ Group #2812 #3289 #2812 #3289#2812 #3289 Filtered 2.02 9.62   97% 95.2%   59% 90% Medium Unfiltered2.13 9.40 96.5% 95.9% 60.2% 89% Medium

The performance of the TIL cultured in the non-filtered medium wassimilar to that of the TIL cultured in the filtered medium. The resultssuggest that particulate serum components have little or no detrimentaleffects on TIL cell growth.

Example 11

This example demonstrates that culturing TIL in cell medium that lacksbeta-mercaptoethanol (BME) has little or no detrimental effects on TILgrowth or potency.

TIL were cultured in complete medium (CM) (50 mL) with or without BME.After 2-3 weeks, population increase, viability, and % CD3+CD8+ weremeasured. The results are shown in Table 8.×10⁷

TABLE 8 TIL# Harvest Viability CD3CD8 Tumor # of Cells Day; (Harvest(Harvest # Group Seeded D17-29) Ratio Day) Day) 3520 +BME 1.80 × 10⁷2.72 × 10⁷ 1.51 93.40% 59.60% −BME 1.80 × 10⁷ 2.11 × 10⁷ 1.17 93.30%31.60% 3522 +BME 3.00 × 10⁷ 4.18 × 10⁷ 1.39 94.80% 51.40% −BME 3.00 ×10⁷ 4.39 × 10⁷ 1.46 95.10% 48.70% 3523 +BME 3.00 × 10⁷ 6.75 × 10⁷ 2.2595.50% 33.90% −BME 3.00 × 10⁷ 6.90 × 10⁷ 2.30 93.60% 37.60% 3524 +BME1.80 × 10⁷ 1.17 × 10⁸ 6.52 94.50% 91.10% −BME 1.80 × 10⁷ 1.12 × 10⁸ 6.2395.00% 86.50%

The performance of the TIL cultured in the medium without BME wassimilar to that of the TIL cultured in the medium with BME.

Potency of the TIL is measured and compared for TIL cultured in mediumwith or without BME. Effector (TIL) cells (1×10⁵) are co-cultured withtarget (antigen-presenting tumor cells) cells (1×10⁵), with AK1700 usedas a negative control and DM5 A2 used as a positive control. IFN-gammasecretion is measured by ELISA. There was no significant difference inIFN-gamma secretion for the TIL cultured in medium without BME ascompared to TIL cultured in medium with BME. Thus, the absence of BMEfrom the medium has little or no detrimental effects on TIL cell growthor potency.

Example 12

This example demonstrates that feeding the TIL no more frequently thanevery third or fourth day during expansion of the number of TIL in a gaspermeable container has little or no detrimental effects on TIL growth.

TIL were rapidly expanded in a 500 mL gas permeable container (G-Rex100)as described above and fed as described in Table 9. At the end of 14days, fold expansion, viability, and composition were measured.

TABLE 9 Day 14 Original Day 7 Day 9 Day 11 Day 13 (total) Standard 100ml +100 ml +50 ml +100 ml +50 ml 400 ml (Fed every other day) Modified100 ml +150 ml — +150 ml — 400 ml (Fed every third or fourth Day

There was no difference in the fold expansion observed for TIL fed everythird or fourth day as compared to TIL fed every other day. Viabilityand cell composition are set forth in Table 10.

TABLE 10 Viability Phenotyping (%) Sample Group (%) CD3 CD3CD4 CD3CD8CD56 #2761 Standard 98.1 98.4 40.4 49.3 0.6 Modified 97.7 98.6 39.9 51.80.5 #3522 Standard 98.9 96.8 29.9 59.9 1.6 Modified 98.4 97.0 28.8 60.21.5 #3523 Standard 96.1 98.3 20.0 72.3 0.2 Modified 95.7 98.4 20.2 72.10.2 #3524 Standard 92.7 99.1 1.5 95.5 0.1 Modified 90.2 99.3 1.4 95.20.1

Potency of the TIL is measured and compared for TIL cultured and fedevery third or fourth day as compared to TIL fed every other day.Effector cells (1×10⁵) are co-cultured with target cells (1×10⁵), withAK1700 used as a negative control and DM5 A2 used as a positive control.IFN-gamma secretion is measured by ELISA. There was no significantdifference in IFN-gamma secretion for the TIL fed every third or fourthday as compared to TIL fed every other day. Thus, the reduction infeeding frequency has little or no detrimental effects on TIL potency.

There was no difference in viability or cell composition observed forTIL fed every third of fourth day as compared to TIL fed every otherday.

Example 13

This example demonstrates that using no more than one type of cellculture medium for expanding the number of TIL has little or nodetrimental effects on TIL growth.

TIL were rapidly expanded as described above using no more than one typeof cell culture medium or two types of cell culture medium as set forthin Table 11.

TABLE 11 Day 0 Day 5 Day 7 Day 11 Standard (2 50% RPMI 50% RPMI 100%100% medium types) 50% AIM-V 50% AIM-V AIM-V AIM-V + 5% Serum + 5%Serum + 5% Serum + GlutaMAX + Hepes + Hepes + GlutaMAX + IL-2 IL-2 +IL-2 IL-2 OKT3 Modified (No 100% 100% 100% 100% more than 1 AIM-V AIM-VAIM-V AIM-V + medium type) 5% Serum + 5% Serum + 5% Serum + GlutaMAX +GlutaMAX + GlutaMAX + GlutaMAX + IL-2 IL-2 + IL-2 IL-2 OKT3

There was no difference in the fold expansion observed for TIL expandedusing no more than one type of cell culture medium as compared to TILexpanded using two types of cell culture medium. Viability and cellcomposition are set forth in Table 12.

TABLE 12 Phenotyping NT (not tested) Sample Group Viability CD3 CD3CD4CD3CD8 CD56 3524 50/50 96.5% 98.40%  1.80% 94.20% 0.30% AIM-V 97.0%98.10%  1.70% 92.00% 0.30% 3546 50/50 97.7% NT NT NT NT AIM-V 96.8% NTNT NT NT 3552 50/50 96.2% 95.60% 21.30% 70.50% 0.20% AIM-V 97.3% 94.80%24.80% 65.80% 0.20%

There was no difference in the viability or cell composition observedfor TIL expanded using no more than one type of cell culture medium ascompared to TIL expanded using two types of cell culture medium.

Potency of the TIL is measured and compared for TIL expanded using nomore than one type of medium and TIL expanded using two types of medium.Effector cells (1×10⁵) are co-cultured with target cells (1×10⁵), withAK1700 used as a negative control and DM5 A2 used as a positive control.IFN-gamma secretion is measured by ELISA. There was no significantdifference in IFN-gamma secretion for TIL expanded using no more thanone type of cell medium as compared to TIL expanded using two types ofcell medium.

Example 14

This example demonstrates that expanding the number of TIL using ahigher ratio of feeders provides a higher number of TIL.

TIL (5×10⁶) from two tumor samples were expanded as described aboveusing allogeneic feeder cells at a TIL:Feeder cell ratio of 1:100, 1:50,or 1:25. Cells were counted on days 7, 11, and 14. Viability andcellular composition were evaluated on day 14.

There was no difference in cell number between the three groups at Day7. At Days 11 and 14, however, expansion using a higher ratio of feedercells corresponds with a better expansion of the number of TIL (9.9×10⁹TIL at 1:100 ratio; 8×10⁹ at 1:50 ratio, and 5.8×10⁹ at a 1:25 ratio atDay 14 for Sample 1 and 1.0×10¹⁰ TIL at 1:100 ratio; 1.0×10¹⁰ at 1:50ratio, and 8.0×10⁹ at a 1:25 ratio at Day 14 for Sample 2).

Example 15

This example demonstrates that TIL cultured in a gas permeable containerhave the same or better potency as compared to TIL cultured in non-gaspermeable, 24 well plates.

TIL are cultured in a gas permeable container (40 mL, G-Rex10) or in anon-gas permeable, 24-well plate. Effector cells (1×10⁵) are co-culturedwith target cells (1×10⁵), with AK1700 used as a negative control andDM5 A2 used as a positive control. IFN-gamma secretion is measured byELISA. The results are shown in Table 13.

TABLE 13 Melanoma Cell Line A2− A2+ Tumor Samples 888 938 526 6242976-1,2 2998 3524 3552 3560 3561 3556 None A1, 24 A1, 24 A2, 3 A2, 3A3, 31 A2 A2, 25 A1, 2 A2, 23 A1, 24 A1, 66 None 0 0 0 0 0 12 10 0 0 0 00 AK1700-3 1 0 0 89 249 85 >2495 177 45 113 1 239 DM5 22 1011 >1903 >2527 66 >1207 1275 161 118 3 73 3556 24 90 294 93 149 100525 >1811 409 157 310 NT >1541 well plate 3556 G- 87 230 88 129 110308 >1494 340 338 388 NT >1577 Rex10 3552 24 114 70 142 >1295 >1763 184621 627 139 149 NT 212 well plate 3552 G- 81 388 >1699 >2239 >2470363 >1773 >1452 282 307 NT 256 Rex10 3560 24 135 94 110 196 372 173 346484 175 318 NT >1008 well plate 3560 G- 104 168 141 483 624 240 870 453212 572 NT >1004 Rex10 3561 24 70 539 305 166 327 456 402 205 97 264 207252 well plate 3561 G- 84 1215 419 113 216 515 323 265 128 329 253 290Rex10

There was no significant difference in IFN-gamma secretion for TILcultured in gas permeable containers as compared to TIL cultured innon-gas permeable 24 well plates.

Example 16

This example demonstrates the treatment of melanoma using TIL preparedby initially culturing the TIL in gas permeable flasks and then rapidlyexpanding the number of TIL in gas permeable flasks. Tumor tissuesamples were obtained from seven melanoma patients. The tumor tissuesamples were cultured in G-Rex10 flasks. TIL were obtained from thecultured tumor tissue samples. The numbers of TIL were expanded inG-Rex100 flasks. The expanded cells were administered to the patients.

Seven patients were treated. One patient (1) had an objective response(OR) by Response Evaluation Criteria In Solid Tumors (RECIST) standards.Six patients (6) were non-responders (NR).

Example 17

This example demonstrates the treatment of melanoma using TIL preparedby expanding the number of selected TIL in gas permeable flasks.

Tumor tissue samples were obtained from 55 melanoma patients. Competemedium (2 ml) (supplemented with 6000 IU/ml IL-2) was added to the wellsin the top row of each 24-well plate. A single fragment of tumor wasadded to each media-containing well.

TIL were obtained from the tumor tissue samples and cultured as follows.Fragments were incubated in multiwell plates in a humidified incubatorat 37° C., with 5% CO₂ in air for 5 days without disturbance. After 5days, the TIL cultures in plates were monitored for growth by viewingwith an inverted light microscope. At this point TIL and other celltypes have extravasated from the fragment and/or propagated in thewells. Half of the CM in plates was replaced with fresh CM containingIL-2 (6000 IU/ml). Media (1 ml) was aspirated, taking care not todisturb the cells on the bottom of the well, and replaced with 1 ml offresh medium containing IL-2 (6000 CU/ml). Every 2-3 days, orapproximately 3 times per week, the plates were monitored for TILgrowth. When TIL expansion was evident, a sample from the well wascounted to quantify cell concentration. When the culture exceeded 1×10⁶lymphocytes/ml or became nearly confluent then the well was split 1:2.Splitting was accomplished by mixing gently with a transfer pipette andtransferring 1 ml of culture to a new well, then adding 1 ml of CMcontaining IL-2 (6000 IU/ml) to each daughter well. Fragment culturesthat showed growth, up to 24 in total, were split in to 2 wells. Thefirst 12 fragment cultures that required a second split in to 4 wellswere maintained. The remaining fragment cultures were frozen as a pool 1(PF1). Typically, 8 fragment cultures were carried through to an 8 wellsplit and maintained for therapy. The remaining cultures were frozen aspool 2 (PF2). The fragments that were being maintained for therapy wereanalyzed by FACS for CD3, CD4, CD8 and CD56 content as close to the dateof REP as possible.

The cultures were screened for specificity by co-culturing 100 μl of TILwith media only, autologous fresh tumor cells, or autologous fresh tumorcells and MHC Class I antibody, and IFN-γ release was measured. Reactivecultures were selected for expansion.

The numbers of TIL (from the selected cultures that released 200 pg/mlor more of IFN-γ) were expanded in G-Rex100 flasks. The expanded cellswere administered to the patients.

Out of fifty-five patients treated, 18 experienced tumor regression ofgreater than 30% and 24 did not experience tumor regression of at least30%. The treatment outcome of the remaining 13 patients has not yet beenevaluated.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of obtaining an expanded number of TIL from a mammal foradoptive cell immunotherapy comprising: obtaining a tumor tissue samplefrom the mammal; culturing the tumor tissue sample in a first gaspermeable container containing cell medium therein; obtaining tumorinfiltrating lymphocytes (TIL) from the tumor tissue sample; expandingthe number of TIL in a second gas permeable container containing cellmedium therein using irradiated allogeneic feeder cells and/orirradiated autologous feeder cells.
 2. A method of promoting regressionof cancer in a mammal comprising: obtaining a tumor tissue sample fromthe mammal; culturing the tumor tissue sample in a first gas permeablecontainer containing cell medium therein; obtaining TIL from the tumortissue sample; expanding the number of TIL in a second gas permeablecontainer containing cell medium therein using irradiated allogeneicfeeder cells and/or irradiated autologous feeder cells; andadministering the expanded number of TIL to the mammal.
 3. A method ofobtaining an expanded number of TIL from a mammal for adoptive cellimmunotherapy comprising: obtaining a tumor tissue sample from themammal; obtaining TIL from the tumor tissue sample; expanding the numberof TIL in a gas permeable container containing cell medium therein usingirradiated allogeneic feeder cells and/or irradiated autologous feedercells.
 4. A method of promoting regression of cancer in a mammalcomprising: obtaining a tumor tissue sample from the mammal; obtainingTIL from the tumor tissue sample; expanding the number of TIL in a gaspermeable container containing cell medium therein using irradiatedallogeneic feeder cells and/or irradiated autologous feeder cells; andadministering the expanded number of TIL to the mammal.
 5. The method ofclaim 3, further comprising selecting TIL capable of lysing cancercells.
 6. The method of claim 1, wherein culturing the tumor tissuesample comprises culturing the tumor tissue sample submerged under aheight of at least about 1.3 cm of cell culture medium in the first gaspermeable container.
 7. The method of claim 1, wherein culturing thetumor tissue sample comprises culturing the tumor tissue samplesubmerged under a height of at least about 2.0 cm of cell culture mediumin the first gas permeable container.
 8. The method of claim 1, whereinculturing the tumor tissue sample comprises culturing the tumor tissuesample in a gas permeable container having at least about 650 cm² ofcell culture surface area.
 9. The method of claim 1, wherein the canceris melanoma.
 10. The method of claim 1, wherein expanding the number ofTIL comprises culturing the TIL submerged under a height of at leastabout 1.3 cm of cell culture medium in the second gas permeablecontainer.
 11. The method of claim 1, wherein expanding the number ofTIL comprises culturing the TIL submerged under a height of at leastabout 2.0 cm of cell culture medium in the second gas permeablecontainer.
 12. The method of claim 1, wherein expanding the number ofTIL comprises culturing the TIL in a gas permeable container having atleast about 650 cm² of cell culture surface area.
 13. The method ofclaim 1, wherein expanding the number of TIL comprises increasing thenumber of TIL by at least about 1000-fold.
 14. The method of claim 1,comprising expanding the number of TIL in about 5,000 mL to about 10,000mL of cell medium.
 15. The method of claim 1, wherein expanding thenumber of TIL uses about 1×10⁹ to about 4×10⁹ allogeneic feeder cellsand/or irradiated autologous feeder cells.
 16. The method of claim 1,wherein the cell medium in the first and/or second gas permeablecontainer is unfiltered.
 17. The method of claim 1, wherein the cellmedium in the first and/or second gas permeable container lacksbeta-mercaptoethanol (BME).
 18. The method of claim 1, wherein expandingthe number of TIL comprises feeding the cells every no more frequentlythan every third or fourth day.
 19. The method of claim 1, wherein thetumor tissue sample is a melanoma tumor tissue sample.
 20. The method ofclaim 1, wherein expanding the number of TIL uses no more than one typeof cell culture medium.
 21. The method of claim 1, wherein the mammal isa human.
 22. The method of claim 1, wherein expanding the number of TILuses irradiated allogeneic feeder cells and/or irradiated autologousfeeder cells at a ratio of about 1 TIL to at least about 20 feedercells.
 23. The method of claim 1, wherein expanding the number of TILuses irradiated allogeneic feeder cells and/or irradiated autologousfeeder cells at a ratio of about 1 TIL to at least about 50 feedercells.
 24. The method of claim 1, wherein expanding the number of TILuses irradiated allogeneic feeder cells and/or irradiated autologousfeeder cells at a ratio of about 1 TIL to at least about 100 feedercells.